Formation of the embryonic organizer is restricted by the competitive influences of Fgf signaling and the SoxB1 transcription factors.

Kuo CL, Lam CM, Hewitt JE, Scotting PJ - PLoS ONE (2013)

Bottom Line:
We show that ectopic expression of organizer genes induced solely by the inhibition of SoxB1 function is dependent upon the activation of fgf expression.These data allow us to describe a comprehensive signaling network in which the SoxB1 factors restrict organizer formation by inhibiting Fgf, Nodal and Wnt signaling, as well as independently repressing the targets of that signaling.The organizer therefore forms only where Nodal-induced Fgf signaling overlaps with Wnt signaling and the SoxB1 proteins are absent.

Affiliation: Centre for Genetics and Genomics, School of Biology, University of Nottingham, QMC, Nottingham, United Kingdom.

ABSTRACTThe organizer is one of the earliest structures to be established during vertebrate development and is crucial to subsequent patterning of the embryo. We have previously shown that the SoxB1 transcription factor, Sox3, plays a central role as a transcriptional repressor of zebrafish organizer gene expression. Recent data suggest that Fgf signaling has a positive influence on organizer formation, but its role remains to be fully elucidated. In order to better understand how Fgf signaling fits into the complex regulatory network that determines when and where the organizer forms, the relationship between the positive effects of Fgf signaling and the repressive effects of the SoxB1 factors must be resolved. This study demonstrates that both fgf3 and fgf8 are required for expression of the organizer genes, gsc and chd, and that SoxB1 factors (Sox3, and the zebrafish specific factors, Sox19a and Sox19b) can repress the expression of both fgf3 and fgf8. However, we also find that these SoxB1 factors inhibit the expression of gsc and chd independently of their repression of fgf expression. We show that ectopic expression of organizer genes induced solely by the inhibition of SoxB1 function is dependent upon the activation of fgf expression. These data allow us to describe a comprehensive signaling network in which the SoxB1 factors restrict organizer formation by inhibiting Fgf, Nodal and Wnt signaling, as well as independently repressing the targets of that signaling. The organizer therefore forms only where Nodal-induced Fgf signaling overlaps with Wnt signaling and the SoxB1 proteins are absent.

pone-0057698-g001: SoxB1 factors acts as transcriptional repressors to inhibit the expression of fgf3 and fgf8.At 4.5 hpf, the expression of fgf3 and fgf8 is restricted in the dorsal shield region of un-injected embryos (A–B). Injection of sox3, sox19a or sox19b RNA at the 1–2 cell stage caused complete loss of expression of both fgf3 and fgf8 at 4.5 hpf (C–H). A Sox3HMG-EnR (I,J) but not a Sox3HMG-VP16 (K,L) fusion mimicked the function of wild-type Sox3 to inhibit fgf3 and fgf8 expression. Ectopic expression of fgf3 and fgf8 was induced by the dnSox3 construct injected at the 1–2 cell stage and analysed at 4.5 hpf (M,N) or later at 30% epiboly (5.5 hpf) (O,P). All images are lateral views with dorsal to the right (where this can be determined). The proportion of embryos exhibiting these phenotypes is shown at the bottom right of each panel. Scale bar in panel A represents approximately 100 µm.

Mentions:
We first examined whether members of the SoxB1 subfamily could affect expression of these fgfs during the period of organizer induction. Injection of sox3, sox19a or sox19b RNA at the 1–2 cell stage resulted in a complete loss of expression of both fgf3 and fgf8 in the organizer (Fig. 1A–H). We found that RNA encoding a Sox3HMG-enR fusion protein, but not the constitutive activator Sox3HMG -VP16, was able to mimic the ability of the SoxB1 factors to repress the expression of both fgf3 and fgf8 (Fig. 1I–L). Hence, it appears that the SoxB1 factors are likely to repress the fgf genes directly.

pone-0057698-g001: SoxB1 factors acts as transcriptional repressors to inhibit the expression of fgf3 and fgf8.At 4.5 hpf, the expression of fgf3 and fgf8 is restricted in the dorsal shield region of un-injected embryos (A–B). Injection of sox3, sox19a or sox19b RNA at the 1–2 cell stage caused complete loss of expression of both fgf3 and fgf8 at 4.5 hpf (C–H). A Sox3HMG-EnR (I,J) but not a Sox3HMG-VP16 (K,L) fusion mimicked the function of wild-type Sox3 to inhibit fgf3 and fgf8 expression. Ectopic expression of fgf3 and fgf8 was induced by the dnSox3 construct injected at the 1–2 cell stage and analysed at 4.5 hpf (M,N) or later at 30% epiboly (5.5 hpf) (O,P). All images are lateral views with dorsal to the right (where this can be determined). The proportion of embryos exhibiting these phenotypes is shown at the bottom right of each panel. Scale bar in panel A represents approximately 100 µm.

Mentions:
We first examined whether members of the SoxB1 subfamily could affect expression of these fgfs during the period of organizer induction. Injection of sox3, sox19a or sox19b RNA at the 1–2 cell stage resulted in a complete loss of expression of both fgf3 and fgf8 in the organizer (Fig. 1A–H). We found that RNA encoding a Sox3HMG-enR fusion protein, but not the constitutive activator Sox3HMG -VP16, was able to mimic the ability of the SoxB1 factors to repress the expression of both fgf3 and fgf8 (Fig. 1I–L). Hence, it appears that the SoxB1 factors are likely to repress the fgf genes directly.

Bottom Line:
We show that ectopic expression of organizer genes induced solely by the inhibition of SoxB1 function is dependent upon the activation of fgf expression.These data allow us to describe a comprehensive signaling network in which the SoxB1 factors restrict organizer formation by inhibiting Fgf, Nodal and Wnt signaling, as well as independently repressing the targets of that signaling.The organizer therefore forms only where Nodal-induced Fgf signaling overlaps with Wnt signaling and the SoxB1 proteins are absent.

Affiliation:
Centre for Genetics and Genomics, School of Biology, University of Nottingham, QMC, Nottingham, United Kingdom.

ABSTRACTThe organizer is one of the earliest structures to be established during vertebrate development and is crucial to subsequent patterning of the embryo. We have previously shown that the SoxB1 transcription factor, Sox3, plays a central role as a transcriptional repressor of zebrafish organizer gene expression. Recent data suggest that Fgf signaling has a positive influence on organizer formation, but its role remains to be fully elucidated. In order to better understand how Fgf signaling fits into the complex regulatory network that determines when and where the organizer forms, the relationship between the positive effects of Fgf signaling and the repressive effects of the SoxB1 factors must be resolved. This study demonstrates that both fgf3 and fgf8 are required for expression of the organizer genes, gsc and chd, and that SoxB1 factors (Sox3, and the zebrafish specific factors, Sox19a and Sox19b) can repress the expression of both fgf3 and fgf8. However, we also find that these SoxB1 factors inhibit the expression of gsc and chd independently of their repression of fgf expression. We show that ectopic expression of organizer genes induced solely by the inhibition of SoxB1 function is dependent upon the activation of fgf expression. These data allow us to describe a comprehensive signaling network in which the SoxB1 factors restrict organizer formation by inhibiting Fgf, Nodal and Wnt signaling, as well as independently repressing the targets of that signaling. The organizer therefore forms only where Nodal-induced Fgf signaling overlaps with Wnt signaling and the SoxB1 proteins are absent.